This invention relates to the field of microorganism detection. In particular, this invention provides an improved methodology for detecting chitinous contaminants in samples, e.g. biological samples.
A methodology for rapid detection and identification of microorganisms, and other contaminants, has long been a concern to the medical, pharmaceutical and food processing fields, among others. Because of this sustained interest, significant advances over the classical time consuming methods of plate counting, membrane filtration, or multiple tube fermentation procedures have been noted. Such approaches include, differential dye-cell wall binding, mass spectrometry, bacteriophage lysis, computer assisted probability methods, gel ferrography, flow cytometry, and the like. Such methods, however, typically have not achieved industry acceptance particularly in the agricultural and food industry. This lack of acceptance/industry implementation is due to disadvantages such as laborious and time-consuming laboratory preparation and sample handling, long observation times and nonspecificity with respect to microorganism characterization and subsequent identification, and increased expense in terms of labor and/or instrumentation. Thus, relatively primitive methods are employed for monitoring contaminants, e.g. in agricultural products.
Thus, for example, California tomato industry monitors mold levels in raw product at inspection stations and in processed product in quality control laboratories. At the inspection stations, 23 kg of fruit from each 24 metric ton truckload of processing tomatoes are visually inspected for defects. Tomatoes with visible signs of mold are weighed to obtain a percentage of decayed fruit on a mass basis (PTAB, 1996). At the processor""s quality control laboratories, mold is quantified by the Howard mold count (HMC) method (AOAC, 1984), where a small drop of homogenized juice is inspected using a microscope. In the HMC method, two slides of twenty-five fields each are viewed, and the percentage of fields containing mold are recorded. An accurate HMC takes up to thirty minutes to conduct.
Over the last several decades, many attempts have been made to replace manual grading and the Howard mold count with a less subjective and less labor-intensive measurement, but no method has been accurate, rapid, and simple enough to use at inspection stations or in quality control laboratories (Jarvis and Williams (1987). p. 599-636. In Food and Beverage Mycology, ed. L. R. Beuchat, 2nd ed. Van Nostrand Reinhold, N.Y.; Gourama and Bullerman (1995) Journal of Food Protection 58:1389-1394; and Cousin (1996) Journal of Food Protection, 59: 73-81). Despite the difficulties and limitations of the HMC (e.g. Williams (1968) J. Ass. Pub. Analysts, 6: 69-84; Jarvis et al. (1983) J. Appl. Bacteriol. 55: 325), it remains the universal standard for mold assessment almost ninety years after it was first introduced (Howard (1911) U.S. Dept. Agr., Bureau of Chemistry, Circular No. 68).
Chitin is an important structural component in fungal cell walls, but absent from plant tissue. The detection of mold based on the chemical isolation and quantification of N-acetyl-D-glucosamine, a breakdown product of chitin, has been proposed as an alternative mold measurement (Ride and Drysdale (1972) Physiol. Plant. Pathol. 2: 7-15; Jarvis (1977) J. Food Technol. 12: 581-591; Lin and Cousin (1985) Journal of Food Protection, 59: 73-81). Jarvis (1977), supra., found a coefficient of variation (CV) of around 20% for this method. Although the high performance liquid chromatography based isolation method is too slow and labor intensive to be utilized as an industrial replacement for the HMC, a more rapid method that detects chitin could have commercial promise (Cousin (1996) Journal of Food Protection, 59: 73-81).
Lectins are naturally occurring proteins or glycoproteins that bind to specific carbohydrates. They are becoming increasingly valuable as molecular probes, including the labeling of cell-surface components in tissue typing (Lis and Sharon (1986) Ann. Rev. Biochem. 55: 35-67). Hundreds of lectins from microbial, plant, and animal cells have been identified, but most commercially available lectins are isolated from plant seeds. They are available with various enzymatic and fluorescent labels, and their nomenclature derives from the name of their source.
There are numerous commercially available lectins that bind polymers of N-acetyl-D-glucosamine. Stoddard and Herbertson (1978) J. Med. Microbiol. 11: 315-324, utilized fluorescein labeled lectins to detect human pathogenic fungi. Patel (1992) The applications of lectins in food analysis. Trends in Food Sci. and Technol. 3: 35-39, used fluorescein isothiocyanate (FITC) labeled lectins to observe mold in processed foods. He tested several chitin-binding lectins, and found that a lectin from wheat germ agglutinin (WGA) had the strongest binding to fungal cell walls and the least amount of nonspecific binding to tomato cells. He observed considerable autofluorescent signal, tomato cell tissue that fluoresces at similar wavelengths as the fluorescent probe. Patel et al. (1993) pages 31-41 In New Techniques in Food and Beverage Microbiology Eds. Kroll, R. G., Gilmour, A., and Sussman, M. Blackwell Science Inc. Oxford, England, used biotinylated lectins and streptavidin labeled magnetic particles to separate and concentrate mold spores and yeasts in fruit juices.
This invention provides novel methods for the detection of chitinous contaminants of non-chitinous biological materials. The methods are accurate, highly reproducible, rapid and relatively inexpensive. The methods are well suited to commercial applications, particular in the food and agriculture industry where biological materials (e.g. food products) are regularly screened for contaminants (e.g. insect, mold, fungus, etc.).
In one embodiment this invention provides a method of detecting chitinous material in a processed non-chitinous biological sample. The method involves contacting the biological sample with a probe that is a lectin that binds chitin, contacting the biological sample with a pectinase, and detecting binding of the lectin to a chitin wherein the binding indicates the presence of chitin (and hence a chitinous contaminant) in the biological sample. The chitinous contaminant can be any of a wide variety of contaminants including, but not limited to insects, insect parts, other animals or parts of animals of the phylum arthropoda (e.g. crustacea), nematodes, annelids, molds, fungi, slimes, yeasts, and various other microorganisms, and the like. In particularly preferred embodiments, the detected contaminant is a fungus of phylum Ascomycota, Basidomycota, Chytridiomycota, or Zygomycota, or a member of the phylum Oomycota in the Stramenopila kingdom. Particularly preferred fungi include, but are not limited to Cladosporium spp, Fusarium spp, Stemphylium spp, Alternaria spp, Geotrichum spp, Fusarium spp, Rhizopus spp, Botrytis spp, Phytophthora spp, Pythium spp, or Pythium spp (e.g. Cladosporium herbarum, Fusarium oxysporum, and Stemphylium botryosum, Alternaria alternata, Geotrichum candidum, Fusarium oxysporum, Rhizopus stolonifer, Botrytis cinerea, Phytophthora parasitica, Pythium aphanidermatum, Pythium ultimum, etc.).
Preferred biological samples include, but are not limited to an agricultural product, a food product, a wood product, a textile, and an animal tissue product. Particularly preferred agricultural products include, but are not limited to fruits, vegetables, grains, forages, silages, juices (vegetable or fruit), a wood, flowers, or seeds. In one embodiment the agricultural product is a tomato, a pepper, a grape, an apple, an orange, a lemon, a berry, or a juice or concentrate thereof.
Preferred lectins for use in this invention include, but are not limited to wheat germ agglutenin (WGA), succinylated WGA, pokeweed lectin, tomato lectin, potato lectin barley lectin, rice lectin, stinging nettle lectin, a vicilin, a chitovibrin, a Vibrio lectin, and a hevein. The lectin is preferably a lectin labeled with a detectable label (e.g., a radioactive label, a magnetic label, a colorimetric label, an enzymatic label, a fluorescent label, a metal, an antibody, a biotin, an avidin, or streptavidin). Fluorescent labels are most preferred. Where fluorescent labels are used, the detecting preferably involves using a fluorometer to detect fluorescence of the label. In particularly preferred embodiments the fluorometer uses a bandpass filter. While virtually any fluorometer is suitable in a most preferred embodiment, the fluorometer is a surface-reading fluorometer.
In certain embodiments, the method is performed at a basic pH greater than about pH 7 (e.g. at about pH 8).
Preferred pectinases for use in the methods of this invention include, but are not limited to polygalacturonases, pectinesterases, pectin lyases, and hemicellulases. The methods of this invention also, optionally, involve using a blocking agent to reduce non-specific binding of the lectin. Blocking agents are well known to those of skill in the art. One preferred blocking agent is serum albumin (e.g. bovine serum albumin).
Preferred processed biological samples include samples that have been subjected to an operation selected from the group consisting of comminuting, homogenizing, heating, evaporation, lyophylization, filtering, concentrating, filtering, fermenting, freezing, and blanching.
In certain embodiments, the biological sample is selected from the group consisting of a fruit, a vegetable, a fruit juice, and a vegetable juice, the lectin is a fluorescently labeled lectin selected from the group consisting of wheat germ agglutenin (WGA), succinylated WGA, pokeweed lectin, tomato lectin, potato lectin barley lectin, rice lectin, stinging nettle lectin, a vicilin, a chitovibrin, a Vibrio lectin, and a hevein, the pectinase is a pectinase selected from the group consisting of polygalacturonases, pectinesterases, pectin lyases and hemicellulases, the sample is processed by comminuting, homogenizing, heating, evaporation, lyophylization, filtering, concentrating, filtering, fermenting, freezing, and blanching; and the detecting comprises detecting a signal from the fluorescent label labeling said lectin.
In another embodiment, this invention provides methods of detecting chitinous material in a non-chitinous biological sample. These methods involve contacting the biological sample with a fluorescently labeled probe that is a lectin that binds chitin, where the contacting is in a solution at a pH ranging from about pH 7 to about pH 9, contacting said biological sample with a fluorescently labeled probe that is a lectin that binds chitin; and detecting binding of the lectin to a chitin wherein said binding indicates the presence of chitin in said biological sample. Preferred contaminants, biological samples, and lectins include one or more of the biological samples, lectins, and contaminants described above. Preferred detection methods utilize a fluorometer (e.g. a surface-reading fluorometer) to detect fluorescence of said label. The fluorometer is optionally equipped with a bandpass filter. The assay is preferably performed at a pH greater than about pH 7.5, more preferably at a pH about pH 8.0. In one preferred embodiment, the biological sample is a fruit, a vegetable, a fruit juice, or a vegetable juice, the lectin is a fluorescently labeled lectin selected from the group consisting of wheat germ agglutenin (WGA), succinylated WGA, pokeweed lectin, tomato lectin, potato lectin, barley lectin, rice lectin, stinging nettle lectin, a vicilin, a chitovibrin, a Vibrio lectin, and a hevein, and the detecting comprises detecting a signal from the fluorescent label labeling said lectin. The method may optionally further involve contacting the biological sample with a pectinase (e.g., a polygalacturonase, a pectinesterase, a pectin lyase a hemicellulase, etc.).
In another embodiment this invention provides a biological sample in which a lectin that specifically binds to chitin is bound to a chitinous contaminant of the sample, and the lectin is labeled with a label that provides a signal distinguishable from a background signal where the signal indicates the presence or quantity of the chintinous contaminant in the biological sample. Such a biological sample may occur in an assay of this invention or may be utilized as a positive control. In preferred embodiments ,the pH of the sample is basic ranging from about pH 7 to about pH 9. Preferred samples are processed samples e.g., as described herein. The sample may, optionally, further comprise an exogenously supplied pectinase. Preferred lectins and/or pectinases include, but are not limited to lectins and/or pectinases as described herein.
In still another embodiment, this invention provides a kit for detecting chitinous material in a non-chitinous biological sample. Preferred kits include a first container containing a lectin that specifically binds a chitinous material, and a second container containing a pectinase. In certain embodiments, the first container and second containers are the same container. Pectinases and/or lectins include, but are not limited to the pectinases and/or the lectins described herein. Certain kits can, optionally, further comprise a label for labeling the lectin, while in certain other embodiments, the lectin is already labeled. The kit can optionally further comprise a transparent centrifugable receptacle (e.g. a flow-through centrifuge) suitable for use with a surface-reading fluorometer and/or a bandpass filter for that passes light emitted by a fluorescent label in the kit.
In yet another embodiment, this invention. provides methods of detecting a fluorochrome bound to one phase of a two-phase mixture. These methods involve contacting a transparent surface of a receptacle with a solid or semi-solid phase of the two phase mixture, illuminating the solid or semisolid phase of the two phase mixture through the transparent surface, and detecting through the transparent surface fluorochrome bound to the solid or semi-solid phase of said two-phase mixture. The receptacle is preferably a centrifuge tube (e.g. or a flow-through centrifuge). The contacting can comprise spinning the receptacle so that the solid or semi-solid phase is deposited against the transparent surface. In preferred embodiments, the two-phase mixture comprises a biological sample (e.g. as described above), with preferred two-phase mixtures including, but not being limited to, fruit and/or vegetable juices, homogenates, or concentrates). The fluorochrome is preferably a chitin-specific fluorescently labeled lectin as described herein.
This invention also provides a surface-reading fluorometer comprising a receptacle having a transparent surface, the receptacle being compatible with centrifugation in a centrifuge; a light source for illuminating a sample through the transparent surface; and a detector disposed to detect fluorescence through the transparent surface.
The terms xe2x80x9cchitinous materialxe2x80x9d refers to a material comprising chitin and/or a breakdown product of chitin (e.g. N-acetyl-D-glucosamine). Chitin includes regenerated chitin (fully acetylated chitin) and chitosan (deacetylated chitin).
The term xe2x80x9clectinsxe2x80x9d refers to carbohydrate-binding proteins or glycoproteins of non-immune origin that agglutinate cells or that precipitate glycoconjugates, or are simply carbohydrate-binding proteins of non-immune origin. Lectins have been isolated from a wide variety of organisms, including bacteria, invertebrates, vertebrates, and plants. Lectins are often glycosylated, and are frequently composed of homo- or heterodimers with one binding site per subunit.
The term xe2x80x9cspecifically bindsxe2x80x9d, when referring to the interaction of a lectin probe and its chitin target refer to a binding reaction that is determinative of the presence of the chitin (or chitin degradation product) in a heterogeneous population of molecules (e.g., proteins and other biologics). Thus, for example, in the case of a lectin of this invention, the lectin preferentially (or specifically) binds to chitin (or chitin degradation product) when the chitin-lectin complex can be distinguished from interactions between the lectin and the non-chitinous biological sample. In general, a signal to noise ratio of 1.2 or greater preferably 1.5 or greater, more preferably 2 or greater, and most preferably 3 or greater (where the signal to noise is the ratio of chitin-specific signal to background signal) indicates detection of a specific binding.
A xe2x80x9cprocessed samplexe2x80x9d or a xe2x80x9cprocessed biological samplexe2x80x9d refers to a sample of a biological material subjected to one or more processes typically used in the commercial preparation of the biological material. Processing often involves one or more forms of preservation, and/or disinfection, and/or fractionation, and/or homogenization or other xe2x80x9clyticxe2x80x9d process. Thus, for example, processing may involve extraction of a juice from a fruit or a vegetable and/or pasturization of that juice. Other xe2x80x9cprocessingxe2x80x9d operations are well known to those of skill in the art and include, but are not limited to addition of preservatives, exposure to radiation, lyophilization, crystallization, dehydration, comminution, heating (e.g. pasturization), evaporation, lyophylization, filtration, concentration, fermentation, freezing, and blanching.
A xe2x80x9cnon-chitinous biological samplexe2x80x9d refers to a biological sample taken from an organism, an organ, and/or a tissue, and/or a cell or cell culture in which chitin does not typically exist. Examples of xe2x80x9cnon-chitinous biological samplesxe2x80x9d include, but are not limited to samples of higher plants (e.g. fruits) or products derived therefrom (e.g. juices, juice concentrates, homogenates, etc.) samples from vertebrates, and the like.
Pectinases are well known enzymes that characteristically break down pectins (polysaccharides typically found in plant cell walls). The term xe2x80x9cpectinasesxe2x80x9d is intended to include polygalacturonases (EC3.2.1.15), pectinesterases (EC3.2.1.11), pectin lyases (EC4.2.2.10) and hemicellulases such as endo-1,3-xcex2-xylosidase (EC 3.2.1.32), xylan 1,4-xcex2-xylosidase (EC 3.2.1.37), xcex1-L-arabinofuranosidase (EC 3.2.1.55), and the like.
A xe2x80x9cmicroorganismxe2x80x9d generally refers to a living organism too small to be seen with the naked eye. Microorganisms include, but are not limited to bacteria, fungi, protozoans, microscopic algae, and viruses.
xe2x80x9cAgricultural product(s)xe2x80x9d, as used herein, refers to plants and/or plant parts, more preferably commercially relevant plants and/or plant parts. Such plants and/or plant parts include, but are not limited to plant vegetative organs (e.g. leaves, roots, stems, and tubers), flowers and floral organs (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), wood and cellulose-based products, seed (including embryo, endosperm, and seed coat), and fruit (the mature ovary), e.g. the harvested product of numerous agronomically-important crop plants. Agricultural products also include processed agricultural products (e.g. dried fruits and/or vegetables, fruit and/or vegetable homogenates and/or concentrates, and/or juices, textiles or cotton products, wood products, etc.) Preferred crop plants include, but are not limited to tomatoes, citrus fruits, pears, apples, peaches, corn, oats, wheat, rice, soybean, alfalfa, barley, millet, hops, as well as numerous grains and/or forages.